Abstract

Mutations of comparative gene identification 58 (CGI-58) in humans cause Chanarin-Dorfman syndrome, a rare autosomal recessive
disease in which excess triacylglycerol (TAG) accumulates in multiple tissues. CGI-58 recently has been ascribed two distinct
biochemical activities, including coactivation of adipose triglyceride lipase and acylation of lysophosphatidic acid (LPA).
It is noteworthy that both the substrate (LPA) and the product (phosphatidic acid) of the LPA acyltransferase reaction are
well-known signaling lipids. Therefore, we hypothesized that CGI-58 is involved in generating lipid mediators that regulate
TAG metabolism and insulin sensitivity. Here, we show that CGI-58 is required for the generation of signaling lipids in response
to inflammatory stimuli and that lipid second messengers generated by CGI-58 play a critical role in maintaining the balance
between inflammation and insulin action. Furthermore, we show that CGI-58 is necessary for maximal TH1 cytokine signaling in the liver. This novel role for CGI-58 in cytokine signaling may explain why diminished CGI-58 expression
causes severe hepatic lipid accumulation yet paradoxically improves hepatic insulin action. Collectively, these findings establish
that CGI-58 provides a novel source of signaling lipids. These findings contribute insight into the basic mechanisms linking
TH1 cytokine signaling to nutrient metabolism.

Comparative gene identification 58 (CGI-58), also known as α/β hydrolase domain-containing protein 5 (ABHD5), recently has
gained attention as the master regulator of triacylglycerol (TAG) hydrolysis and phospholipid metabolism (1–4). However, molecular mechanisms by which CGI-58 regulates these metabolic processes still are incompletely understood. Because
the discovery that mutations in CGI-58 cause Chanarin-Dorfman syndrome (CDS) (5), several groups have studied CGI-58’s biochemical properties in vitro (1–4). An important advancement on this front came when it was demonstrated that CGI-58 indirectly promotes TAG hydrolysis by
coactivating adipose triglyceride lipase (ATGL) (1). However, recent studies in mice with diminished levels of CGI-58 clearly show that ATGL-independent functions for CGI-58
also must exist (2,6). In addition to activating ATGL, CGI-58 catalyzes the acylation of lysophosphatidic acid (LPA) to generate the critical
lipid second messenger phosphatidic acid (PA). Both the substrate (LPA) and the product (PA) of the LPA acyltransferase (LPAAT)
reaction are well-known signaling lipids with critical roles in angiogenesis, cardiac development, carcinogenesis, and immunity
(7–9). Furthermore, fibroblasts from CDS patients have dramatically altered rates of synthesis and turnover of other major lipids
with signaling potential, including phosphatidylcholine (PC), phosphatidylinositol, and phosphatidylserine (10,11). Given the central importance of lipid mediators in growth factor and cytokine-mediated signal transduction (7–9), we reasoned that CGI-58 may be a novel source of signaling lipids. Unfortunately, conventional gene targeting of CGI-58
in mice results in premature lethality (6). To circumvent this, we used targeted antisense oligonucleotides (ASOs) to test whether CGI-58 plays a quantitatively important
role in the generation of signaling lipids in vivo. Our findings show that CGI-58 is a novel source of signaling lipids that
links inflammation to TAG and glucose metabolism.

RESEARCH DESIGN AND METHODS

Male C57BL/6N mice (Harlan) were maintained on standard rodent chow or a high-fat diet (HFD) for a period of 4–10 weeks and
simultaneously injected with ASOs targeting knockdown (KD) of CGI-58, as previously described (2). The diets and ASOs used here have been described elsewhere (2). The HFD was prepared by our institutional diet core and contains ~45% of energy as lard (16:0 = 23.3, 18:0 = 15.9, 18:1
= 34.8, and 18:2 = 18.7%). The 20-mer phosphorothioate ASOs were designed to contain 2'-0-methoxyethyl groups at positions
1–5 and 15–20 and were synthesized, screened, and purified, as described previously (12), by ISIS Pharmaceuticals (Carlsbad, CA). The CGI-58 ASOs used in the current studies were described as CGI-58 ASOβ in our previous work (2). All mice were maintained in an American Association for Accreditation of Laboratory Animal Care–approved specific pathogen-free
environment on a 12:12-h light:dark cycle and allowed free access to regular chow and water. All experiments were performed
with the approval of the institutional animal care and use committee.

Lipopolysaccharide-induced acute-phase response.

Mice were injected with control or CGI-58 ASOs and maintained on standard chow or an HFD for a period of 4 weeks, as previously
described (2). After 4 weeks of ASO treatment, mice were injected intraperitoneally with either saline or 5 μg lipopolysaccharide (LPS)
(Escherichia coli 0111:B4). Following injection, plasma was collected at 1 h by submandibular puncture (for tumor necrosis factor [TNF] α measurements),
and exactly 6 h after injection mice were killed with ketamine/xylazine (100–160 mg/kg ketamine and 20–32 mg/kg xylazine).
Thereafter, a midline laparotomy was performed, and blood was collected by heart puncture. After blood collection, a whole-body
perfusion was conducted by puncturing the inferior vena cava and slowly delivering 10 mL sterile 0.9% saline into the left
ventricle of the heart to remove residual blood. Multiple tissues were collected and snap-frozen for subsequent analysis.

In vivo insulin-signaling analyses.

Mice were injected with control or CGI-58 ASOs and maintained on standard chow or an HFD for a period of 8 weeks, as previously
described (2). After an overnight fast (11:00 p.m. to 9:00 a.m.), mice were anesthetized with isoflurane (4% for induction and 2% for maintenance) and were maintained on a 37°C heating
pad to control body temperature. A minimal midline laparotomy was performed, and the portal vein was visualized. Sterile saline
or recombinant human insulin (0.5 units/kg body wt; Novo Nordisk) was administered directly into the portal vein. Exactly
5 min later, tissues were excised without saline perfusion and immediately snap frozen in liquid nitrogen. Protein extracts
from tissues were analyzed by Western blotting, as previously described (13–15).

In vivo hepatic TNFα-signaling analyses.

Mice were injected with control or CGI-58 ASOs and maintained on standard chow or an HFD for a period of 4 weeks, as previously
described (2). After an overnight fast (11:00 p.m. to 9:00 a.m.), mice were anesthetized with isoflurane (4% for induction and 2% for maintenance) and were maintained on a 37°C heating
pad to control body temperature. A minimal midline laparotomy was performed, and the portal vein was visualized. Saline or
mouse recombinant TNFα (10 ng/mouse, no. 410-MT; R&D Systems) was administered directly into the portal vein. Exactly 5 min
later, the liver was excised without saline perfusion and immediately snap-frozen in liquid nitrogen. Protein extracts from
tissues were analyzed by Western blotting, as previously described (13–15), and lipid extracts were analyzed using mass spectrometry methods (16), as described in detail below.

Plasma biochemistries.

Detailed descriptions of plasma lipid and lipoprotein analyses have been previously described (14,15). Plasma cytokines were quantified by multiplex assay (Bio-Plex; Bio-Rad) as previously described (18). In some cases (Fig. 4A), the level of plasma TNFα was determined by enzyme-linked immunosorbent assay (no. MTA00; R&D Systems).

LPA acyltransferase activity assay.

Whole-liver homogenates were prepared from snap-frozen mouse liver by dounce homogenization in 50 mmol/L Tris, pH 7.5; 300
mmol/L NaCl; 50 mmol/L NaF; and Sigma protease inhibitor cocktail just before the assay. Cellular debris was removed by centrifugation
at 1,000g for 15 min at 4°C, and the supernatant was used for determination of protein content by Lowry assay (19). Total hepatic LPAAT activity was determined by measuring the conversion of [1-14C]-oleoyl-CoA to [1-14C]-PA, as previously described (3). The reaction was assembled in 100 μL of 50 mmol/L Tris (pH 7.5) containing 50 μmol/L oleoyl-sn1-glycerol-3-phosphate (Avanti Polar Lipids, Alabaster, AL) and 10 μmol/L [1-14C]-oleoyl-CoA (specific activity 8,000 dpm/nmol) using 5 μg of whole-liver homogenate and continued for 10 min at 30°C.

Immunoblotting.

Whole-tissue homogenates were made from multiple tissues in a modified radioimmunoprecipitation assay buffer, as previously
described (13–15). Proteins were separated by 4–12% SDS-PAGE and transferred to polyvinylidene difluoride membranes, and proteins were detected
after incubation with specific antibodies. Information on the antibodies used is available upon request.

Hepatic neutral lipid and glycerophospholipid analyses.

Extraction of liver lipids for enzymatic quantification of total TAG, cholesteryl esters (14,15), free cholesterol, and phospholipid was performed as previously described. Glycerophospholipids were extracted using a modified
Bligh and Dyer procedure (20). Approximately 10 mg of frozen mouse liver was homogenized in 800 μL ice-cold 0.1 N HCl:CH3OH (1:1) using a tight-fit glass homogenizer (Kimble/Kontes Glass, Vineland, NJ) for ~1 min on ice. Suspension was then transferred
to cold 1.5-mL microfuge tubes (Laboratory Product Sales, Rochester, NY) and vortexed with 400 μL cold CHCl3 for 1 min. The extraction proceeded with centrifugation (5 min, 4°C, 18,000g) to separate the two phases. The lower organic layer was collected and the solvent was evaporated. The resulting lipid film
was dissolved in 100 μL isopropanol:hexane:100 mmol/L NH4CO2H(aq) (58:40:2) (mobile phase A). Quantification of glycerophospholipids was achieved by the use of a liquid chromatography–mass
spectrometry technique using synthetic (non–naturally occurring) diacyl and lysophospholipid standards. Typically, 200 ng
of each odd-carbon standard was added per 10–20 mg tissue. Glycerophospholipids were analyzed on an Applied Biosystems/MDS
SCIEX 4000 Q TRAP hybrid triple quadrupole/linear ion trap mass spectrometer (Applied Biosystems, Foster City, CA) and a Shimadzu
high-pressure liquid chromatography system with a Phenomenex Luna Silica column (2 × 250 mm, 5-μm particle size) using a gradient
elution, as previously described (16). The identification of the individual species, achieved by liquid chromatography–tandem mass spectrometry, was based on
their chromatographic and mass spectral characteristic. This analysis allows identification of the two fatty acid moieties
but does not determine their position on the glycerol backbone (sn-1 vs. sn-2). TAG, diacylglycerol (DAG), and monoacylglycerol (MAG) from frozen mouse liver tissue (10–15 mg) were extracted by homogenizing
tissue in the presence of internal standards (500 ng each of 14:0 MAG, 24:0 DAG, and 42:0 TAG) in 2 mL 1× PBS and extracting
with 2 mL ethyl acetate:trimethylpentane (25:75). After drying the extracts, the lipid film was dissolved in 1 mL hexane:isopropanol
(4:1) and passed through a bed of Silicagel 60 Å to remove the remaining polar phospholipids. Solvent from the collected fractions
was evaporated and lipid film was redissolved in 100 μL CH3OH:CHCl3 (9:1), containing 10 μL of 100 mmol/L CH3COONa for mass spectrometry analysis, essentially as described previously (21).·

Quantitative real-time PCR.

Tissue RNA extraction and quantitative real-time PCR (qPCR) was conducted as previously described (14,15). Cyclophilin or hypoxanthine phosphoribosyltransferase 1 was used as invariant controls for these studies, and expression
levels were calculated based on the ΔΔCT method. qPCR was conducted using the Applied Biosystems 7500 Real-Time PCR System. Primers used for qPCR are available on
request.

Statistical analysis.

All data are expressed as means ± SEM and were analyzed using either a one-way or two-way ANOVA followed by Student t tests for post hoc analysis using JMP version 5.0.12 software (SAS Institute, Inc., Cary, NC).

RESULTS

CGI-58 KD paradoxically improves hepatic insulin signaling.

Our original interest in CGI-58’s role in intracellular signaling was sparked by the unexplained “metabolic paradox” apparent
in mice with diminished CGI-58 function (Fig. 1). We have discovered that CGI-58 KD results in striking hepatic steatosis (Fig. 1A) (2) yet paradoxically improves systemic glucose and insulin tolerance (2). It is well accepted that hepatic lipotoxicity, and more specifically the hepatic accumulation of signaling lipids such
as DAG and ceramides, is linked to insulin resistance (22). However, hepatic lipid insult is not sufficient to cause insulin resistance in CGI-58 ASO-treated mice (Fig. 1) (2). Instead, CGI-58 KD actually improves systemic insulin action despite these metabolic abnormalities (2). To confirm that the hepatic steatosis seen in CGI-58 ASO-treated mice were indeed dissociated from primary defects in insulin
signaling, we analyzed acute Akt and FoxO1 phosphorylation in response to portally administered insulin (Fig. 1B–G). In agreement with previous measures of systemic glucose and insulin tolerance (2), CGI-58 KD significantly improved hepatic insulin signaling (Fig. 1B and E). CGI-58 KD had no significant impact on insulin-stimulated Akt phosphorylation in skeletal muscle (Fig. 1C and F) and adipose tissue (Fig. 1D and G). Collectively, these data have uncovered an unexpected role for CGI-58 in dissociating hepatic steatosis from insulin resistance.
This prompted us to examine the molecular basis for this dissociation.

CGI-58–generated signaling lipids are necessary for maximal TNFα signaling in the liver. A–C: Mice were maintained on a chow diet for 4 weeks in conjunction with biweekly injections (25 mg/kg) of either a nontargeting
control ASO (□) or ASO targeting knockdown of CGI-58 (CGI-58 ASO; ■). Mice were fasted for 10 h before injection of saline
or TNFα (10 ng) into the portal vein. Exactly 5 min later, the liver was excised and immediately snap-frozen in liquid nitrogen
for signaling analyses. A: Hepatic levels of PA and phosphatidylglycerol (PG) were analyzed by mass spectrometry. B: Total hepatic LPAAT activity. Data in A and B represent the mean ± SEM from four mice per group, and values not sharing a common superscript letter differ significantly
(P < 0.05). C: Protein extracts from the liver were analyzed for total IκB α (IκBα) and phospho-IκBα (p-IκBα; Ser32); data from four representative
animals are shown for each group. D–F: Acute stress kinase activation in primary hepatocytes. Following 4 weeks of ASO treatment, hepatocytes were isolated from
control and CGI-58 ASO-treated mice by collagenase perfusion. Freshly isolated hepatocytes were stimulated for 15 min (15’)
or 1 h with 100 ng/mL TNFα (D), 10 ng/mL IL-1β (E), or 10 ng/mL IL-6 (F). Downstream signaling was analyzed by immunoblotting for p-JNK (Thr183/Tyr185), phospho-S6 ribosomal protein (p-S6; Ser235/236),
and β-actin. Data in D–F represent responses of hepatocytes isolated from three individual mice per condition.

CGI-58 KD alters the systemic response to endotoxin.

Although TH1 cytokines (TNFα, IL-1β, and IL-6) clearly have been implicated in promoting chronic inflammatory conditions that accompany
obesity (23–28), TH1 cytokine action has been best characterized in models of acute inflammation driven by microbial infection or tissue injury
(36,37). In acute inflammation, TH1cytokines are produced transiently by macrophages and mast cells to promote tissue reprogramming typified by the hepatic
acute-phase response (34,36,37). Of interest, the acute-phase response in the liver also is associated with transient overproduction of VLDL-TAG (34) and insulin resistance (24,25), two pathways that are stimulated by CGI-58 in vivo (2) (Figs. 1D–F). To test whether CGI-58 participates in cytokine action and TAG metabolism during acute inflammation, we injected CGI-58
ASO-treated mice with a low dose of LPS (E. coli). In response to LPS, CGI-58 KD significantly elevated plasma levels of TNFα, IL-6, and IL-12p40, compared with LPS-injected
controls (Fig. 4A). Of note, 1 h after LPS injection, mice that received CGI-58 ASOs had fivefold more circulating TNFα than LPS-injected control
mice (Fig. 4A). Despite the elevation in circulating TH1 cytokines (Fig. 4A), LPS-induced expression of TNFα and markers of the acute-phase response (serum amyloid A [SAA] and serum amyloid P-component
[SAP]) were significantly reduced in livers of CGI-58 ASO-treated mice (Fig. 4B). LPS-induced plasma levels of SAA and haptoglobin also were decreased in CGI-58 ASO-treated mice (Fig. 4A). However, LPS induced higher expression of other cytokines, such as IL-12p40 and IL-10, in the livers of CGI-58 ASO-treated
mice (Fig. 4B). It is noteworthy that, in parallel to altered circulating levels of cytokines (Fig. 4A), LPS injection increased the expression of TNFα, IL-6, IL12p40, and IL-10 in adipose of CGI-58 ASO-treated mice (Fig. 4C).

Of note, the LPS response in white adipose tissue–treated mice was unique in CGI-58 ASO-treated mice (Supplementary Fig. 11). In support of this, LPS-induced expression of IL-1β, TNFα, and several other TH1 cytokines was dramatically elevated in the white adipose tissue of CGI-58 ASO-treated mice compared with that of control
mice (Fig. 4C and Supplementary Fig. 11; data not shown). In contrast, LPS-driven expression of IL-1β and TNFα was reciprocally diminished in the liver, lung, spleen,
and kidney of CGI-58 ASO-treated mice compared with LPS-injected controls (Supplementary Fig. 11). Both white and brown adipose tissue from CGI-58 ASO-treated mice had four- to sevenfold higher expression of the macrophage
marker CD-68 (Supplementary Fig. 11).

We surmised that the integrated inflammatory response to endotoxin was dramatically altered by CGI-58 KD (Fig. 4 and Supplementary Fig. 11). However, we were concerned that this effect may be simply a result of the abnormally high accumulation of TAGs in the liver
of CGI-58 ASO-treated mice (2) (Supplementary Fig. 1). To rule out this possibility, we fed mice an HFD for 4 weeks, which increased hepatic TAG levels to the same levels seen
in chow-fed CGI-58 ASO-treated mice (Supplementary Fig. 12B). We then treated these HFD-fed mice with LPS to determine whether HFD-induced fatty liver could alter the acute-phase response
in a similar fashion to CGI-58 ASO treatment. Of importance, HFD feeding did not mimic the effects of CGI-58 KD on LPS-driven
plasma cytokine levels (Supplementary Fig. 12C and D) or the hepatic acute-phase response (Supplementary Fig. 12E). Moreover, CGI-58 ASO treatment increased plasma TH1 cytokines and blunted the acute-phase response of mice on both chow and HFDs (Supplementary Fig. 12C–E), further supporting the idea that CGI-58 ASO-driven alteration in inflammatory signaling is an on-target effect of the ASO
and not a result of hepatic TAG accumulation.

Given CGI-58’s documented role in promoting adipose lipolysis (1) and hepatic VLDL-TAG packaging (2), we examined these parameters in LPS-injected, CGI-58 ASO-treated mice. LPS treatment increased plasma nonesterified fatty
acid levels by 18% in chow-fed control ASO-treated mice and 25% in chow-fed CGI-58 ASO-treated mice, indicating that LPS-driven
adipose lipolysis was similar between groups (data not shown). However, in these same mice, the hepatic metabolic response
to LPS was altered (Fig. 4D and E). LPS treatment of chow-fed CGI-58 ASO-treated mice resulted in a significant (29%) increase in hepatic TAG levels (Fig. 4D) but no comparable change in control ASO-treated mice. Of interest, LPS treatment caused a 73% increase in plasma TAG levels
in chow-fed control ASO-treated mice yet caused no hypertriglyceridemia in CGI-58 ASO-treated mice (Fig. 4E). These data suggest that hepatic CGI-58 plays a critical role in the overproduction of TAG-rich lipoproteins during infection.
Collectively, these data suggest that CGI-58 function is critical to both the inflammatory and metabolic response to acute
infection.

DISCUSSION

Although it generally is accepted that CGI-58 indirectly regulates TAG metabolism by coactivating ATGL (1), we now alternatively propose that CGI-58’s ability to acylate LPA (3,4) also plays a critical role in CGI-58’s ability to modulate TAG metabolism and insulin signaling. The major findings of the
current study are that CGI-58 KD in mice 1) improves insulin signaling in liver and skeletal muscle; 2) prevents HFD-induced stress kinase activation; 3) prevents the generation of PA and other glycerophospholipid species in response to TNFα, thereby attenuating downstream
signaling; and 4) alters the integrated inflammatory response to endotoxin. In our current working model (Fig. 5), we propose that downstream of hepatic cytokine receptor activation, in response to inflammatory stimuli such as an HFD
or LPS treatment, CGI-58 generates signaling lipids either directly through direct acylation of LPA or indirectly by coactivating
ATGL-mediated TAG hydrolysis. CGI-58–generated PA, and likely other signaling lipids, can subsequently act as lipid second
messengers to activate stress kinases such IKK-β, S6K1, and mTOR. These stress kinases can then facilitate serine phosphorylation
of critical residues on IRS-1, thereby dampening hepatic insulin signaling (Fig. 5). This role in cytokine signaling may partially explain why CGI-58 KD causes severe hepatic lipid insult and yet improves
hepatic insulin signaling.

Proposed model for CGI-58’s integrated role in inflammatory responses and insulin action in the liver. In response to inflammatory
stimuli, such as an HFD or LPS, plasma levels of inflammatory cytokines, such TNFα, IL-1β, and IL-6, are increased. These
inflammatory cytokines normally signal through their membrane-bound receptors (TNF-R, IL-6-R, and IL-1-R) to promote CGI-58–driven
generation of signaling lipids either directly from LPAAT activity or indirectly by coactivating ATGL to generate lipid signals
from TAG hydrolysis. CGI-58–generated PA, and likely other signaling lipids, can subsequently act as a critical second messenger
to promote the activation of inflammatory stress kinases, such as IKK-β, S6K1, and mTOR. Collectively, these activated stress
kinases (IKK-β, S6K1, and mTOR) can facilitate serine phosphorylation (pS) of critical serine residues (Ser307, Ser612, Ser632,
and Ser1101) on IRS-1, thereby dampening hepatic insulin signaling. Knocking down CGI-58 diminishes this potent negative regulatory
loop, thereby improving hepatic insulin action. IR, insulin receptor. TNF-R, TNF receptor.

It has now been a decade since the causal link between CGI-58 mutations and CDS was established (5), yet molecular mechanism(s) by which CGI-58 prevents CDS has remained elusive. Early studies using skin fibroblasts isolated
from patients with neutral lipid storage disease or CDS showed that these cultured cells had striking accumulation of intracellular
TAGs under normal growth conditions (10,11,38–41). However, the TAG accumulation could not be explained by alteration in mitochondrial fatty acid uptake, β-oxidation, in
vitro lipase activity, or TAG synthesizing enzyme activity (10,11,38–41). Instead, it was found that neutral lipid storage disease fibroblasts had impaired turnover of long-chain fatty acids from
stored TAGs (38–41). We have likewise demonstrated that targeted knockdown of CGI-58 in hepatocytes impairs intracellular TAG hydrolysis in
vitro and in vivo (2,42). Of interest, CGI-58 is a lipid-droplet–associated protein in adipocytes, achieving this subcellular localization by directly
interacting with perilipin A (43,44). However, it is important to note that CGI-58 is not always associated with lipid droplets in nonadipocyte cell models (42–44), and the intracellular trafficking itinerary of CGI-58 under hormonal or cytokine stimulation deserves further study.

The product of the LPAAT reaction, PA, is a well-studied signaling lipid (7–9,45–47). In fact, PA participates in many cellular signal transduction pathways and regulates membrane trafficking (7–9,45–47). It is generally accepted that PA regulates cell signaling by physically interacting with target proteins through defined
PA-binding motifs, thereby altering either membrane localization or activation state. Bona fide PA-binding proteins include
protein kinases, phosphatases, phosphodiesterases, scaffolding proteins, and small guanine nucleotide exchange factors (7–9,45–47). Although the majority of acute cytokine-stimulated PA generation has been attributed to the enzymatic hydrolysis of PC
through the action of phospholipase D (45–47) or the phosphorylation of DAGs by DAG kinases (48), there is growing evidence that LPAAT enzymes make substantial contributions to endotoxin- and cytokine-stimulated PA generation
(29–33). In fact, pharmacologic inhibition of LPAAT activity protects mice against endotoxic shock, lung injury, and pancreatic
islet dysfunction in response to endotoxin and IL-1 (32,33,49,50), implicating LPAAT-derived PA in promoting inflammatory disease. Undoubtedly, PA is a central lipid signaling molecule that
can be synthesized or broken down by a number of enzymatic pathways (45–50). We propose that CGI-58–driven synthesis of PA represents a novel lipid-signaling pathway that may have important implications
in human diseases, such as the metabolic syndrome and CDS. CGI-58 KD in mice prevents diet-induced obesity and decreases fat-pad
mass (2), suggesting a defect in lipid storage by adipose tissue. The possibility that CGI-58–generated signaling lipids may regulate
adipocyte function in vivo deserves further investigation. The signaling function of CGI-58 may also have implications for
neurologic defects in CDS, including ataxia, mental retardation, and hearing loss. Given that global deficiency of CGI-58
results in postnatal lethality, tissue-specific CGI-58 knockout mice will be required to further dissect the role of CGI-58–generated
signaling lipids in these other biological processes. In conclusion, these studies demonstrate that CGI-58 is a novel source
of signaling lipids that integrate inflammation and nutrient metabolism.

ACKNOWLEDGMENTS

This work was supported by the Department of Pathology at Wake Forest University and by grants from the American Heart Association
(grant 11BGIA7840072 to J.M.B.); the National Heart, Lung, and Blood Institute (grants 1-K99-HL-096166 to J.M.B., 5-T32-HL-091796
to C.C.L., and 5-P01-HL-049373 to J.S.P.); the National Institute of Diabetes and Digestive and Kidney Diseases (grants 1-F32-DK-084582
to J.L.B. and 1-R01-054797 to D.L.B.); the National Institute of General Medical Sciences LIPID MAPS (grant U54-GM-069338
to H.A.B.); and the Intramural Research Program of the National Institute of Environmental Health Sciences (grant NIEHS Z01-ES-102005
to M.B.F.).

No potential conflicts of interest relevant to this article were reported.

C.C.L., J.L.B., G.T., and M.A.D. conducted the experiments, analyzed the data, and aided in the manuscript preparation. P.T.I.,
S.B.M., D.S.M., and H.A.B. performed all lipidomic analyses and provided critical insights for these studies. S.C., M.L.,
and J.S.P. performed primary hepatocyte isolations. R.G.L., R.M.C., and M.J.G. provided antisense oligonucleotides and valuable
discussion. D.L.B. aided in the measurements of hepatic LPAAT activity. M.B.F. and J.M. performed plasma cytokine analyses
and discussed the data. J.M.B. planned the project, designed the experiments, analyzed the data, wrote the manuscript, and
is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the
integrity of the data and the accuracy of the data analysis. All authors were involved in the editing of the final manuscript.

The authors thank all members of the laboratories of J.M.B. (Wake Forest University School of Medicine), Ryan Temel (Wake
Forest University School of Medicine), Larry Rudel (Wake Forest University School of Medicine), and Paul Dawson (Wake Forest
University School of Medicine) for comments and suggestions. The authors also thank Deanna Russell (Rutgers University) for
technical assistance.